Method for improved measurement of local physical parameters in a fluid-filled cavity

ABSTRACT

The present invention relates to remotely determining local physical parameters in a fluid-filled cavity (e.g. heart cavities, blood vessels, industrial container) by means of ultrasound waves and encapsulated or stabilised gas bubbles. A measuring method, a method of diagnostic ultrasound of the same and an apparatus for remotely determining ambient physical local parameters of a fluid-filled cavity are disclosed.

This application claims the benefit of provisional application60/281,794 filed on Apr. 6, 2001.

TECHNICAL FIELD

The present invention relates to a noninvasive measuring method forremotely determining local physical parameters of a fluid-filled cavity,by means of ultrasound waves and encapsulated or stabilized gas bubbles(e.g. suspensions of stabilized microbubbles, microballoons ormicroparticles comprising gas).

BACKGROUND OF THE INVENTION

Physiological parameters of the cardiovascular system, such as bloodpressure, temperature and gas concentration are important since theyprovide essential information concerning the state of health of organsand the patient. Currently, dynamic blood pressure measurements aremainly performed by catheterization, consisting of a pressure-sensingcatheter that is inserted into the heart chamber or blood vessel, or byDoppler echocardiography using the simplified Bernoulli equation (BurtonC. Physiology and biophysics of the circulation. 2^(nd) edition.Chicago, 1972). The first method is accompanied by the disadvantages ofan invasive procedure, i.e. creating pain and risk of infection. Thesecond, noninvasive method does not provide reliable or reproducibleblood pressure values (Strauss A L, Roth F J, Rieger H. “Noninvasiveassessment of pressure gradients across iliac artery stenoses: duplexand catheter correlative study” J Ultras Med 1993; 12: 17-22).

Alternative techniques are described in the literature and are mainlybased on the interaction of ultrasound waves with individual gas bubbles(Fairbank W, Scully M. “A new noninvasive technique for cardiac pressuremeasurement: resonant scattering of ultrasound from bubbles” IEEE TransBiomed Eng 1977; 24: 107-110; Hök B. “A new approach to noninvasivemanometry: interaction between ultrasound and bubbles” Med Biol EngComput 1981; 19: 35-39; Tickner E G. “Precision microbubbles for rightside intracardiac pressure and flow measurements” Meltzer RS andRoelandt JTCR, ed. Contrast echocardiography; London: Martinus Nijhoff,1982; 15: 313-324; Ishihara K et al. “New approach to noninvasivemanometry based on pressure dependent resonant shift of elasticmicrocapsules in ultrasonic frequency characteristics” Jap J App Phys1988; 27: 125-127; DE 29 46 662 A1 (Siemens AG); EP 0 296 189 B(Schering AG); U.S. Pat. No. 4,483,345 (Miwa).

Due to the high compressibility of gas, the size of a gas bubble changesas a function of the local hydrostatic pressure. This change in sizeaffects the acoustic characteristics of the gas bubble, like resonancefrequency, scattering and attenuation cross-section, etc. Therefore, thelocal pressure in a fluid-filled cavity can be derived from theseacoustic characteristics.

Recent attempts to utilize gas bubbles to noninvasivaly assess thepressure in fluid filled cavities (De Jong et al. WO 98/32378 (AndarisLtd.); Shi et al. WO 99/47045) have been hampered by inaccuracy andinsensitivity

De Jong et al. WO 98/32378 (Andaris Ltd.) and Bouakaz et al.(“Noninvasive measurement of the hydrostatic pressure in a fluid-filledcavity based on the disappearance time of micrometer-sized free gasbubbles” Ultrasound in Medicine and Biology 1999; 25: 1407-1415)disclosed a method for noninvasive measurement of the local pressureinvolving injection of what are referred to as gas containingmicrocapsules into the circulatory system. By transmitting a lowfrequency, high amplitude ultrasound burst, free-gas bubbles arereleased from the gas containing microcapsules into the region where thelocal pressure is to be measured. The disappearance time of the releasedfree gas depends on the local pressure and is used for noninvasivedetermination of the local pressure. In this application, the totalresponse of the released gas bubbles (fundamental and second harmonic)is used to calculate the disappearance time. The Bouakaz article statedthat this method is inaccurate for detecting small pressure changes onthe order of 5-10 mmHg, which are clinically relevant (Bouakaz et al.“Noninvasive measurement of the hydrostatic pressure in a fluid-filledcavity based on the disappearance time of micrometer-sized free gasbubbles” Ultrasound in Medicine and Biology 1999; 25: 1407-1415).

Shi et al. WO 99/47045, stated that an excellent correlation existsbetween the amplitude of subharmonic signals generated by microbubblesand the local pressure. Thus, they suggested that sub and ultraharmonicamplitude may be used to noninvasivaly to estimate the local pressure,asserting that subharmonic amplitudes are a much better indicator ofpressure variation than fundamental and second harmonic amplitudes.However, the difference in sub- and/or ultraharmonic amplitude is verysmall for pressure changes ranging from 5-10 mmHg (Shi et al. “Pressuredependence of subharmonic signals from contrast microbubbles” Ultrasoundin Medicine and Biology 1999; 25: 275-283). Therefore, this method isalso lacking sensitivity when detecting small pressure changes.Secondly, this method strongly depends on the size of the bubbles at thelocation where the pressure is to be measured. In the method disclosedin WO99/47045, after injection of the bubbles the size distribution willchange due to lung filtration and microbubble uptake. Therefore, theexact size of the bubbles, and consequently the acoustic characteristicslike sub- and ultraharmonic-response, at the location of interest isunknown.

It is an object of the present invention to provide a new method foraccurate and sensitive, noninvasive measurement of the local physicalparameters in a fluid-filled cavity. With this new method small pressurechanges (5-10 mmHg) can be measured, which is the main limitation of themethods described by aforementioned references. Additionally, unlike themethod disclosed in WO99/47045, in the present method, the size of thebubbles at the location where the pressure is to be measured can bebetter controlled and, therefore, the acoustic characteristics of thebubbles at the site of interest are better specified, which makes thepresent invention more accurate. With reference to WO98/32378, thepresent invention entails shorter acquisition time, which makes it moreefficient and more useful in the clinic.

This new method can provide clinicians with a valuable tool fordetermining the state of health of an organ without the risk ofinfection and with minimal patient discomfort. Moreover, it will bereadily apparent to those skilled in the art that the present inventioncan be used as a general technique for remotely sensing physicalparameters, for example in situations where direct measurement isimpossible or too dangerous.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a technique fordetermining local physical parameters in a fluid-filled cavity byadministering stabilized or encapsulated gas bubbles and monitoring thesub- and/or ultraharmonic response of the free gas bubbles released fromthese stabilized or encapsulated gas bubbles as a function of time.

The stabilized or encapsulated gas bubbles useful in the invention maybe divided into several categories:stabilized gasmicrobubbles,gas-filled microcapsules/microballoons, and gas containingmicroparticles according to the definitions given in for example, in EP554213 and U.S. Pat. No. 5,413,774.

The term “microbubble” specifically designates gas bubbles, insuspension in a liquid preferably also containing surfactants ortensides to control the surface properties and the stability of thebubbles. The term “microcapsule” or “microballoon” designates preferablyair or gas-filled bodies with a material boundary or envelope, i.e. apolymer membrane wall. The term microparticle refers to gas-containingsolid systems, for example microparticles (especially aggregates ofmicroparticles) having gas contained therein or otherwise associatedtherewith (for example being adsorbed on the surface thereof and/orcontained within voids, cavities or pores therein).

Free-gas bubbles, i.e. gas bubbles that are not stabilized by any means(gas bubbles which are neither stabilized by tensides or surfactants norencapsulated by, for example, a polymer or contained in or associatedwith solids) are obtained from these stabilized or encapsulated gasbubbles by the destruction of the stabilized microbubbles, microballoonsor microparticles by, for example, application of one or more ultrasoundpulsed waves. The released free gas bubbles dissolve in the surroundingliquid. Consequently, the bubbles shrink and the sub- and/orultraharmonic response, which is very sensitive to bubble size, changesas a function of time. The time for a gas bubble to completely dissolvein the surrounding liquid is a function of local parameters like gasconcentration, temperature and pressure. Therefore, by monitoring thechanges in sub- and/or ultraharmonic response of a free gas bubble as afunction of time, these parameters can be measured non-invasively.

The sub- and ultraharmonic responses of gas bubbles are very sensitiveto their sizes. Consequently, the sub- and ultraharmonic responses offree gas bubbles are very sensitive to parameters that influence thebubble size. Therefore, by measuring the change in bubble size due togas dissolving in the surrounding liquid, by means of the response ofsub- and/or ultraharmonics as a function of time, an estimation of thelocal pressure or other parameters can be made.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Radius-time curves for a 2.2-μm bubble of different gases inwater calculated by using equation (1).

FIG. 2. Radius-time curves for a 2.2-μm air bubble in water at 0 and 200mmHg overpressure, calculated by using equation (1).

FIG. 3A. Radius-time curves for a 2.2-μm air bubble in water at 0 mmHgoverpressure, calculated by using equation (1).

FIG. 3B. Sub- and ultraharmonic response and associated spectrum of a2.2-μm air bubble, 25 ms after release.

FIG. 3C. Sub- and ultraharmonic response and associated spectrum of a2.2-μm air bubble, 45 ms after release.

FIG. 3D. Sub- and ultraharmonic response and associated spectrum of a2.2-μm air bubble, 60 ms after release.

FIG. 4. Energy-time curves for different overpressures.

DETAILED DESCRIPTION

One aspect of the present invention is to provide a noninvasivemeasuring method for remotely determining local physical parameters of afluid-filled cavity, based on the combined use of encapsulated orstabilized gas bubbles and ultrasound waves, said measuring methodcomprising the steps of:

-   -   a) administering stabilized or encapsulated gas bubbles to a        fluid filled cavity;    -   b) applying with a transducer a first ultrasound pulsed wave or        a train of pulsed waves to the fluid filled cavity to destroy        the stabilized or encapsulated gas bubbles and generate free gas        bubbles    -   c) applying with a transducer a second ultrasound pulsed wave or        train of pulsed waves at a frequency specifically chosen for        exciting the sub- and/or ultraharmonic response of the bubbles    -   d) determining the mean response time.    -   e) determining a value for a local physical parameter on the        basis of the response time of step d)

It should be mentioned that the improved sensitivity for noninvasivemeasurement in a fluid-filled cavity as shown in this invention wouldnot be obtainable by omitting one of the steps previously described.

For a better understanding of the invention, the local physicalparameters considered here are the pressure, the temperature or the gasconcentration.

Surprisingly, it has been found that the essential features of theinvention lie in that the sub- and/or ultraharmonic responses of step b)are monitored as a function of time by successive ultrasound pulses,this function being an indication of the local physical parameter beingmeasured. The method of the present invention provides improved accuracyand efficiency by measuring the sub- and ultraharmonic response as afunction of time.

The first ultrasound pulsed wave or train of pulsed waves is tuned insuch a way (e.g. by adjusting its frequency and/or amplitude) that thesize of the released free-gas bubbles is larger than the subharmonicsize (as defined later in the specification).

Preferably, the frequency and amplitude of the first ultrasound pulsedwave or train of pulsed waves, to optimize gas release, can be chosenindependently of the second ultrasound pulsed wave or train of pulsedwaves, used for monitoring the sub- and/or ultraharmonic response.

Preferably, the frequency and amplitude of the second ultrasound pulsedwave or train of pulsed waves, used for monitoring the sub- and/orultraharmonic response, can be chosen independently of the firstultrasound pulsed wave or train of pulsed waves, to optimize gasrealise.

The transducer used to apply the first ultrasound pulsed wave or trainof pulsed waves can be the same as or distinct from that used forapplying the second ultrasound pulsed wave or train of pulsed waves.

Viewed from a further aspect, the invention provides a method ofdiagnostic ultrasound for determining local physical parameters of afluid-filled cavity in situ, which comprises the steps of:

-   -   a) administering to a subject a fluid agent comprising        encapsulated or stabilised gas bubbles    -   b) applying with a transducer a first ultrasound pulsed wave or        a train of pulsed waves to generate free-gas bubbles    -   c) applying with a transducer a second ultrasound pulsed wave or        train of pulsed waves around a frequency specifically chosen for        exciting the sub- and/or ultraharmonic response of the bubbles    -   d) determining the mean response time    -   e) determining a value for a local physical parameter on the        basis of the response time of step d)        characterised in that the sub- and/or ultraharmonic responses of        step c) are monitored as a function of time by successive        ultrasound wave pulses, this function being an indication of the        physical parameter being measured.

According to this method, said subject is a vertebrate, and said fluidagent containing encapsulated or stabilised gas bubbles is introducedinto the vasculature or into a body cavity of said vertebrate.

Viewed from a further aspect, the invention provides an apparatus forcarrying out the above method of measurement. This apparatus compriseselements found in ultrasound equipment, coupled with a specific softwareto generate the required excitations and monitoring transmit-pulsesequences, plus the signal-processing functions required forinterpreting the observed responses as a function of the local physicalparameters. The apparatus includes for example: timing circuits, atleast one pulse or arbitrary waveform generator with amplifier, at leastone transmit-transducer capable of projecting an ultrasound wave intothe region of interest, at least one receive-transducer sensitive to thereflected ultrasound waves, at least one receiving circuit dedicated tothe amplification and conditioning of the echo signals, analog ordigital circuits dedicated to perform filtering of the echo responsesfrom microbubbles, a Central Processing Unit capable of performing therequired computations and comparisons with look-up tables to derivevalues of the local physical parameters, a memory for storing therequired programs, look-up tables, signal data and computed values, anddisplay means for presenting the computed values in a graphical ortextual form.

In summary, the objective of the present invention relates to the remotemeasurement of local physical parameters in a fluid-filled cavity (e.g.heart cavities, blood vessels, industrial container, etc.) by means ofultrasound waves and encapsulated or stabilized gas bubbles, which areused as a vehicle for delivering free-gas bubbles to a site of interest.Stabilisation or encapsulation of the gas, by means of, for example,stabilizing with a tenside or surfactant, encapsulating a gas bubble bya wall or shell, or associating it with a solid prevents the gas contentfrom rapidly dissolving in the surrounding liquid. At the site ofinterest, where for example the local pressure or other local parameters(like temperature, gas concentration, etc.) are to be measured, thestabilized or encapsulated structure is ruptured by means of ultrasoundwaves and the gas content is subsequently released. The release can beoptimally tuned for releasing known-size free-gas bubbles e.g. byadjusting the frequency and/or the amplitude of the applied ultrasoundwave (Frinking et al. “Scattering properties of encapsulated gas bubblesat high ultrasound pressures” Journ. Acoust. Soc. Am. 1999: 105;1989-1996). Surprisingly, it has been found that free-gas bubbles aremore susceptible to changes in environmental conditions, like changes intemperature, gas concentration or local pressure, compared to stabilizedor encapsulated gas bubbles.

It is known that the radius of a free-gas bubble changes as a functionof time according to: $\begin{matrix}{\frac{\mathbb{d}R}{\mathbb{d}t} = {\frac{D\quad L}{R}\left( \frac{\frac{C_{i}}{C_{0}} - 1 - \frac{2\sigma}{R\quad P_{0}} - \frac{p_{ov}}{P_{0}}}{1 + \frac{4\sigma}{3R\quad P_{0}}} \right)}} & (1)\end{matrix}$where

-   R=radius of the bubbles-   t=time-   D=diffusion constant-   C_(i)/C_(o)=ratio of the dissolved gas concentration to the    saturation concentration-   σ=surface tension-   P₀=atmospheric pressure-   L=Ostwald coefficient-   p_(ov)=overpressure.

The time for a free-gas bubble to completely dissolve in the surroundingliquid, i.e. the disappearance time, depends on the type of gas. This isreflected in equation (1) by the Ostwald coefficient and diffusionconstant of a gas. For example, for a 2.2-μm gas bubble thedisappearance time is 3.2 ms for CO₂, 120.8 ms for air and 2936.6 ms forC₄F₁₀ (FIG. 1).

In a gas-saturated liquid, the dissolution of micron-sized free-gasbubbles is caused by a difference in gas concentration between the gasinside the bubble and inside the liquid due to surface tension andoverpressure. In FIG. 2, for example, the radius-time curve for a 2.2-μmair bubble in water is simulated at values of the overpressure of 0 mmHgand 200 mmHg. It is assumed that temperature and gas concentration areconstant. The disappearance time is 120.9 and 94.1 ms, respectively.

The ultrasound parameters for releasing free gas bubbles are tuned insuch a way (e.g. by adjusting its frequency and/or amplitude) that thesize of the released bubbles is larger than the subharmonic size. Thesubharmonic size of a gas bubble is defined as the radius of the bubblefor which the resonance frequency equals half the transmitted frequency(f₀). After the release of known-size free-gas bubbles, gas dissolvinginto the surrounding liquid, i.e. the bubble size as a function of time,is monitored by means of ultrasound waves.

When the bubble size equals the subharmonic size, the response of thegas bubble to the monitoring ultrasound waves will show the appearanceof subharmonics (1/2f₀, 1/3f₀, 3/4f₀ . . . ) of the transmittedfrequency. Additionally, the response of the gas bubble to theultrasound waves will show the appearance of ultraharmonics (3/2f₀,5/2f₀ . . . ) of the transmitted frequency.

This is advantageously illustrated in FIG. 3, which shows the responseof a 2.2-μm air bubble to an ultrasound wave at different time instantsduring gas dissolution in the surrounding liquid at 0 mmhg overpressure(101.3 kPa). In this particular example, the subharmonic component ischosen to be 1/2f₀ and the ultraharmonic component is chosen to be3/2f₀. FIG. 3A shows the radius-time curve of a 2.2-μm air bubble inwater. The three dots, I, II and III correspond to a bubble size greaterthan subharmonic size, at subharmonic size and smaller than subharmonicsize, respectively. The corresponding time and frequency responses areshown in FIGS. 3B, 3C and 3D for I, II and III, respectively.

Surprisingly, the sub- and ultraharmonic responses of gas bubbles arevery sensitive to their sizes. Consequently, the sub- and ultraharmonicresponses of gas bubbles are very sensitive to parameters that influencethe bubble size. Measuring the change in bubble size due to gasdissolving in the surrounding liquid, by means of the response of sub-and/or ultraharmonics as a function of time, an improved estimation ofthe local pressure or other parameters can be made. It should bementioned that the improved sensitivity for noninvasive measurement in afluid-filled cavity, as shown in this invention, is only obtainable bythe combination of the sub- and/or ultraharmonic response and thedisappearance time of the released gas bubbles.

After the release of a free-gas bubble with a radius larger than thesubharmonic size, the sub- and ultraharmonic response amplitude of thegas bubble will increase until it reaches a maximum and subsequentlydecrease until it disappears. The mean sub- or ultraharmonic responsetime is defined as the mean time (weighted by for example the energy orcorrelation coefficient of the sub- or ultraharmonic response) for sub-or ultraharmonics to appear after the release of the gas from thestabilized or encapsulated gas bubble. In formula form this is:$\begin{matrix}{{\overset{\_}{T} = \frac{\sum\limits_{t = t_{1}}^{t_{2}}\quad{t \times {E(t)}}}{\sum\limits_{t = t_{1}}^{t_{2}}\quad{E(t)}}},} & (2)\end{matrix}$where

-   {overscore (T)}=mean response time-   t=time-   E(t)=sub- or ultraharmonic energy or correlation at time t-   t₁,t₂=time after the release of free-gas bubbles where the sub- or    ultraharmonic energy is half the maximum energy.

As it is known by a person skilled in the art, absolute values of thelocal physical parameters can be derived from the mean response time, ascalculated in equation 2, by comparing the calculated mean response timewith predetermined values listed in calibration or look-up tables.

The sub- and/or ultraharmonic component of the transmit frequency can beobtained by standard signal processing techniques, like digitalband-pass filtering of the time responses. The energy of the sub- and/orultraharmonic components of the transmit frequency as a function of timeis obtained by taking the sum of the squared value of the filteredresponses at each time point. The correlation of the sub- and/orultraharmonic components of the transmit frequency as a function of timeis obtained by taking the correlation between the filtered responses attwo successive time instants, at each time point.

Special care is taken to tune the monitoring ultrasound waves in such away (e.g. by adjusting its frequency and/or amplitude) as to generatesub- and ultraharmonics and to minimize rupture or destruction of theencapsulated gas-filled microparticles.

The suspensions of encapsulated or stabilised gas bubbles useful in thepresent invention may be divided into three categories: stabilizedmicrobubbles; microballoons (also called microcapsules); andmicroparticles. Free-gas bubbles are not included in these categoriessince, due to their rapid dissolution in the surrounding liquid; theyare not stable enough to reliably deliver gas bubbles to the area ofinterest. Interest has accordingly been shown in methods of stabilisinggas bubbles commonly used for echography and other ultrasonic studies,for example using emulsifiers, oils, thickeners or sugars, or byentrapping or encapsulating the gas or a precursor thereof in a varietyof systems.

For the present invention, the encapsulated or stabilized gas bubblesare microbubbles bounded by a very thin envelope involving thesurfactant bound at the gas to liquid interface, microballoons(microcapsules or gas-filled liposomes) bounded by a material envelopemade of organic polymers or biodegradable water insoluble and at roomtemperature solid lipids or microparticles having gas contained thereinor otherwise associated therewith (for example being adsorbed on thesurface thereof and/or contained within voids, cavities or porestherein).

The first category, belonging to the class of microbubbles, specificallydesignates gas bubbles in suspension in a liquid preferably alsocontaining surfactants or tensides to control the surface properties andthe stability of the bubbles.

Preferably the microbubble suspension comprises a surfactant or atenside, such as, for example, a polyoxyethylene-polyoxypropylene blockcopolymer surfactant such as Pluronic® or a polymer surfactant like thatdisclosed in U.S. Pat. No. 5,919,434. More preferably amphipathiccompounds capable of forming stable films in the presence of water (oran aqueous carrier) and gas are used as surfactants in the stabilizedmicrobubbles useful in the invention. Such compounds may include, forexample a film forming lipid. The lipids, synthetic ornaturally-occurring generally amphipathic and biocompatible, usable forpreparing the gas-containing agents used in the present inventioninclude, for example, fatty acids; lysolipids; phospholipids such as:phosphatidylcholine (PC) with both saturated and unsaturated lipids;including phosphatidylcholine such as dioleylphosphatidylcholine;dimyristoylphosphatidylcholine (DMPC),dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine (DLPC);dipalmitoylphosphatidylcholine (DPPC); disteraoylphosphatidylcholine(DSPC); and diarachidonylphospha-tidylcholine (DAPC);phosphatidylethanolamines (PE), such as dioleylphosphatidylethanolamine,dipalmitoylphosphatidylethanolamine (DPPE) anddistearoyl-phosphatidylethanolamine (DSPE); phosphatidylserine (PS) suchas dipalmitoyl phosphatidylserine (DPPS), disteraoylphosphatidylserine(DSPS); phosphatidylglycerols (PG), such asdipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol(DSPG); phosphatidylinositol; sphingolipids such as sphingomyelin;glycolipids such as gangliosides GM1 and GM2; glucolipids; sulfatides;glycosphingolipids; phosphatidic acids as dipaimitoylphosphatidic acid(DPPA) and distearoylphosphatidic acid (DSPA); fatty acids such as:palmitic acid; stearic acid; arachidonic acid; oleic acid; lipidsbearing polymers, such as chitin, hyaluronic acid, polyvinylpirrolidoneor polyethylene glycol (PEG), also referred as “pegylated lipids”, withpreferred lipids bearing polymers including DPPE-PEG (DPPE-PEG), whichrefers to the lipid DPPE having a PEG polymer attached thereto,including, for example, DPPE-PEG2000, which refers to DPPE havingattached thereto a PEG polymer having a mean average molecular weight ofabout 2000; lipids bearing sulfonated mono- di-, oligo- orpolysaccharides; cholesterol, cholesterol sulfate and cholesterolhemisuccinate; tocopherol hemisuccinate; lipids with ether andester-linked fatty acids; polymerized lipids (a wide variety of whichare well known in the art); diacetyl phosphate; dicetyl phosphate;stearylamine; cardiolipin; phosholipids with short chain fatty acids ofabout 6 to about 8 carbons in length; synthetic phospholipids withasymmetric acyl chains, such as, for example, one acyl chain of about 6carbons and another acyl chain of about 12 carbons; ceramides; non-ionicliposomes including niosomes such as polyoxyethylene fatty acid esters,polyoxyethylene fatty alcohols, polyoxyethylene fatty alcohol ethers,polyoxyethylated sorbitan fatty acid esters, glycerol polyethyleneglycol ricinoleate, ethoxylated soybean sterols, ethoxylated castor oil,polyoxyethylene-polyoxypropilene polymers, and polyoxyethylene fattyacid stearates; sterol aliphatic acid esters including cholesterolsulfate, cholesterol butyrate, cholesterol iso-butyrate, cholesterolpalmitate, cholesterol stearate, lanosterol acetate, ergosterolpalinitate, and phytosterol n-butyrate; sterol esters of sugar acidsincluding cholesterol glucoronides, lanosterol glucoronides,7-dehydrocholesterol glucoronide, ergosterol glucoronide, cholesterolgluconate, lanosterol gluconate, and ergosterol gluconate; esters ofsugar acids and alcohols including lauryl glucoronide, stearoylglucoronide, myristoyl glucoronide, lauryl gluconate, myristoylgluconate, and stearoyl gluconate; esters of sugars and aliphatic acidsincluding sucrose laurate, fructose laurate, sucrose palmitate, sucrosestearate, glucuronic acid, gluconic acid and polyuronic acid; saponinsincluding sarsasapogenin, smilagenin, hederagenin, oleanolic acid, anddigitoxigenin; glycerol dilaurate, glycerol trilaurate, glyceroldipaimitate, glycerol and glycerol esters including glyceroltripalmitate, glycerol distearate, glycerol tristearate, glyceroldimyristate, glycerol trimyristate; long chain alcohols includingn-decyl alcohol, lauryl alcohol, myristyl alcohol, cetyl alcohol, andn-octadecyl alcohol;6-(5-cholesten-3β-yloxy)-1-thio-β-D-galactopyranoside;digalactosyldiglyceride;6-(5-cholesten-3β-yloxy)hexyl-6-amino-6-deoxy-1-thio-β-D-galacto-pyranoside;6-(5-cholesten-3β-yloxy)hexyl-6-amino-6-deoxyl-1-thio-β-D-mannopyranoside;12-(((7′-diethylaminocoumarin-3-yl)carbonyl)-methylamino)octadecanoicacid;N-[12-(((7′-diethylaminocoumarin-3-yl)carbonyl)methylamino)octadecanoyl]-2-aminopalmiticacid; N-succinyldioleylphosphatidylethanolamine;1,2-dioleyl-sn-glycerol; 1,2-dipalmitoyl-sn-3-succinylglycerol;1,3-dipalmitoyl-2-succinylglycerol;1-hexadecyl-2-palmitoylglycerophosphoethanolamine andpalmitoylhomocysteine, and/or combinations thereof.

Preferably, the lipid is a film forming phospholipid and more preferablythe film forming phospholipid material may be selected from saturatedphospholipids or synthetic non-saturated phospholipids or a mixturethereof. Examples of suitable phospholipids are saturated syntheticlecithins, such as, dimyristoylphosphatidylcholine,dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine ordiarachidoylphosphatidylcholine or unsaturated synthetic lecithins, suchas dioleylphosphatidyl choline or dilinoleylphosphatidylcholine or mixedchains phosphatidylcholines such as for instancemonooleylmonopalmitoylphosphatidylcholine, with saturated phospholipidsbeing preferred. Even more preferably, the saturated phospholipid may beselected from saturated phosphatidic acid, saturatedphosphatidylcholine, saturated phosphatidyl-ethanolamine, saturatedphosphatidylserine, saturated phosphatidylglycerol, saturatedphosphatidyl-inositol, saturated cardiolipin and saturatedsphingomyelin. Particularly preferred are the saturated phospholipidsselected in the following group: dimyristoylphosphatidylethanolamine,dipalmitoylphosphatidylethanolamine, distearoylphosphatidylethanolamineor diarachidoylphosphatidylethanolamine; ordioleylphosphatidylethanolamine or dilinoleylphosphatidylethanolamine,fluorinated analogues of any of the foregoing, mixtures of any of theforegoing, with saturated phosphatidylcholine being preferred.

Additives like cholesterol and other substances can optionally be addedto one or more of the foregoing lipids in proportions ranging from zeroto 50% by weight. Such additives may include other non-phospholipidsurfactants that can be used in admixture with the film formingsurfactants and most of which are known. For instance compounds likepolyoxypropylene glycol and polyoxyethylene glycol as well as copolymersthereof, ergosterol, phytosterol, sitosterol, lanosterol, tocopherol,propyl gallate, ascorbyl palmitate and butylated hydroxytoluene, fattyacids such as myristic acid, palmitic acid, stearic acid, arachidic acidor their derivatives. Particularly preferred is palmitic acid. Theamount of these non-film-forming surfactants is usually up to 50% byweight of the total amount of surfactants but preferably between 0 and30%.

This category includes aqueous suspensions in which the gas bubbles arebounded at the gas/liquid interface by a very thin layer of surfactantbound at the gas to liquid interface. Easy-to-produce aqueousmicrobubble suspensions usable in the present invention are disclosedin, for example, EP 474833 (U.S. Pat. No. 5,271,928), U.S. Pat. Nos.5,380,519, 5,531,980, 5,567,414, 5,643,553, 5,658,551, 5,911,972,incorporated herein by reference in their entirety. The suspensionscontain film forming surfactants in laminar and/or lamellar form and,optionally, hydrophilic stabilisers. These microbubbles are stabilisedby one or more mono-molecular layer(s) of amphipathic compounds i.e.compounds with hydrophilic and hydrophobic moieties. These patents alsodisclose a dry composition, which, upon admixing with an aqueous carrierliquid, will generate a sterile suspension of microbubbles thereafterusable in the present invention. Preferred suspensions containphospholipids as film forming surfactants and, optionally, hydrophilicstabilizers. The total concentration of phospholipids may range from0.01% to 20% and preferably comprised between 0.01-10% (w/w) of thetotal lipid concentration and even most preferably between 0.1-1% (w/w).The concentration of microbubbles may range from 10⁷ to 10¹⁰ bubbles/mL,with a preferred concentration of is between 10⁸ and 10⁹ bubbles/mL. Themicrobubble suspensions remain stable for months.

Preferred phospholipid monolayer stabilized microbubbles are alsodisclosed in for example, U.S. Pat. Nos. 5,445,813; 5,597,549; 5,686,060(Schneider et al), U.S. Pat. Nos. 5,413,774; 5,578,292 (Schneider etal), U.S. Pat. Nos. 5,556,610; 5,846,518 (Yan et al) and U.S. Pat. No.5,827,504 (Yan et al), incorporated herein by reference in theirentirety.

Other microbubble suspensions useful in the invention include thosedisclosed in, for example, U.S. Pat. No. 5,798,091 (Trevino et al) andWO 97/29783 (Nycomed designating the US, also EP 881 915), incorporatedherein by reference in their entirety.

For example, U.S. Pat. No. 5,798,091 discloses what is stated to be agas emulsion comprising a plurality of bubbles surrounded by a layer ofat least a first and a second surfactant. The first surfactant is ahydrophobic phospholipid or mixture of phospholipids having at least oneacyl chain, which comprises at least 10 carbon atoms, which is at leastabout 5% w/w of the total surfactant. The second surfactant is may ormay not also be a phospholipid or mixture of phospholipids, but which ismore hydrophilic than the phospholipid or combination of phospholipidprovided as the first surfactant. Preferred second surfactants may beselected from the group consisting of phospholipids, phosphocholines,lysophospholipids, nonionic surfactants, neutral or anionic surfactants,fluorinated surfactants, which can be neutral or anionic, andcombinations of such emulsifying or foaming agents. Some specificexamples of surfactants which are useful as the second surfactantinclude block copolymers of polyoxypropylene and polyoxyethylene (anexample of such class of compounds is Pluronic, such as Pluronic F-68),sugar esters, fatty alcohols, aliphatic amine oxides, hyaluronic acidaliphatic esters, hyaluronic acid aliphatic ester salts, dodecylpoly(ethyleneoxy)ethanol, nonylphenoxy poly(ethyleneoxy) ethanol,derivatized starches, hydroxy ethyl starch fatty acid esters, salts offatty acids, commercial food vegetable starches, dextran fatty acidesters, sorbitol fatty acid esters, gelatin, serum albumins, andcombinations thereof. Also contemplated as a second surfactant arepolyoxyethylene fatty acids esters, such as polyoxyethylene stearates,polyoxyethylene fatty alcohol ethers, polyoxyethylated sorbitan fattyacid esters, glycerol polyethylene glycol oxystearate, glycerolpolyethylene glycol ricinoleate, ethoxylated soybean sterols,ethoxylated castor oils, and the hydrogenated derivatives thereof. Inaddition, nonionic alkylglucosides such as Tweens®, Spans® and Brijs®may also be used as the second surfactant.

In WO 9729783, there is provided a contrast agent for use in diagnosticstudies comprising a suspension in an injectable aqueous carrier liquidof gas microbubbles stabilised by phospholipid-containing amphiphilicmaterial characterised in that said amphiphilic material consistsessentially of phospholipid predominantly comprising molecules with netcharges. Desirably at least 75%, preferably substantially all of thephospholipid material in the contrast agents consists of moleculesbearing a net overall charge under conditions of preparation and/or use,which charge may be positive or, more preferably, negative.Representative positively charged phospholipids include esters ofphosphatidic acids such as dipalmitoylphosphatidic acid ordistearoylphosphatidic acid with aminoalcohols such ashydroxyethylenediamine. Examples of negatively charged phospholipidsinclude naturally occurring (e.g. soya bean or egg yolk derived),semisynthetic (e.g. partially or fully hydrogenated) and syntheticphosphatidylserines, phosphatidylglycerols, phosphatidylinositols,phosphatidic acids and cardiolipins. The fatty acyl groups of suchphospholipids will typically each contain about 14-22 carbon atoms, forexample as in palmitoyl and stearoyl groups. Lyso forms of such chargedphospholipids are also useful, the term “lyso” denoting phospholipidscontaining only one fatty acyl group, this preferably being ester-linkedto the 1 position carbon atom of the glyceryl moiety. Such lyso forms ofcharged phospholipids may advantageously be used in admixture withcharged phospholipids containing two fatty acyl groups.

The preparation of a preferred gas-filled microbubble suspension usefulin the invention can be done according to, for example, the methodsdescribed in the following patents: EP 554213; U.S. Pat. Nos. 5,413,774;5,578,292; EP 744962; EP 682530; U.S. Pat. No. 5,556,610; EP 474833;U.S. Pat. Nos. 5,271,928; 5,380,519; 5,531,980; 5,567,414; EP 619743;U.S. Pat. Nos. 5,445,813; 5,597,549, incorporated by reference herein intheir entirety.

Regardless of how the microbubble suspension is prepared, in order topermit free passage through the pulmonary system and to achieveresonance, it may be convenient to employ microbubbles having an averagesize of 0.1-10 μm. Microbubbles used in the present invention may beproduced with a very narrow size distribution for the microbubbledispersion within the range preferred for echography, thereby greatlyenhancing their effective echogenicity as well as their safety in vivo,and rendering the microbubbles of particular advantage in suchapplication.

The second category includes contrast agents with a solid materialenvelope formed of natural or synthetic polymers. In this case, the gasfilled bodies are referred to as microballoons. The term “microballoon”or “microcapsule” designates gas-filled bodies with a material boundaryor envelope, i.e. a polymer membrane wall. Gas-filled liposomesaccording to, for example, U.S. Pat. No. 5,580,575 (Unger) also belongto this category and are incorporated herein by reference. More on thesedifferent formulations may be found in EP-A-0 324 938 (U.S. Pat. No.4,844,882, Widder et al.), U.S. Pat. No. 5,711,933 (Bichon et al.), U.S.Pat. No. 4,900,540 (Ryan), U.S. Pat. No. 5,230,882 (Unger), U.S. Pat.No. 4,718,433 (Feinstein), U.S. Pat. No. 4,774,958 (Feinstein), WO9501187 (MBI designating the US), U.S. Pat. No. 5,529,766 (Nycomed),U.S. Pat. No. 5,536,490 (Nycomed), U.S. Pat. No. 5,990,263 (Nycomed) thecontents of which are incorporated herein by reference.

Microballoons, which may be particularly useful in the presentinvention, include pressure resistant microballoons bounded by a softand elastic membrane, which can temporarily deform under variations ofpressure and are endowed with enhanced echogenicity and arebiodegradable.

For polymeric microballoon, the polymer, which constitutes the envelopeor bounding membrane of the injectable microballoons can be selectedfrom most hydrophilic, biodegradable physiologically compatiblepolymers. Such polymers include polysaccharides of low water solubility,polylactides and polyglycolides and their copolymers, copolymers oflactides and lactones such as e-caprolactone, γ-valerolactone andpolypeptides. Other suitable polymers include poly-(ortho)esters (seefor instance U.S. Pat. Nos. 4,093,709; 4,131,648; 4,138,344; 4,180,646also incorporated herein by reference); polylactic and polyglycolic acidand their copolymers, for instance DEXON (see J. Heller, Biomaterials 1(1980), 51, incorporated herein by reference in its entirety);poly(DL-lactide-co-γ-caprolactone), poly(DL-lactide-co-γ-valerolactone),poly(DL-lactide-co-g-butyrolactone), polyalkylcyanoacrylates;polyamides, polyhydroxybutyrate; polydioxanone; poly-β-aminoketones(Polymer 23 (1982), 1693, incorporated herein by reference in itsentirety); polyphosphazenes (Science 193 (1976), 1214, incorporatedherein by reference in its entirety); and polyanhydrides. References onbiodegradable polymers can be found in R. Langer et al., Macromol. Chem.Phys. C23 (1983), 61-126, incorporated herein by reference in itsentirety. Polyamino-acids such as polyglutamic and polyaspartic acidscan also be used as well as their derivatives, i.e. partial esters withlower alcohols or glycols. One useful example of such polymers ispoly-(t.butyl-glutamate). Copolymers with other amino-acids such asmethionine, leucine, valine, proline, glycine, alamine, etc. are alsopossible. Recently some novel derivatives of polyglutamic andpolyaspartic acid with controlled biodegradability have been reported(see U.S. Pat. Nos. 4,892,733; 4,888,398 and 4,675,381 incorporatedherein by reference in their entirety). Biodegradable water insolubleand at room temperature solid lipids selected from a mono-, di- ortri-glycerides, fatty acids, sterols, waxes and the mixtures thereof mayalso be used for the manufacture of microballoons according to theinvention (see WO 96/15815 incorporated herein by reference in itsentirety). Non-biodegradable polymers for making microballoons can beselected from most water-insoluble, physiologically acceptable,bioresistant polymers including polyolefins (polystyrene), acrylicresins (polyacrylates, polyacrylonitrile), polyesters (polycarbonate),polyurethanes, polyurea and their copolymers. ABS(acryl-butadiene-styrene) is a preferred copolymer.

Properties of the membrane, for instance strength, elasticity andbiodegradability, can be controlled. Additives can be incorporated intothe polymer wall of the microballoons to modify the physical propertiessuch as dispersibility, elasticity and water permeability. Among theuseful additives, one may cite compounds which can “hydrophobize” themicroballoons membrane in order to decrease water permeability, such asfats, waxes and high molecular-weight hydrocarbons. Additives, whichimprove dispersibility of the microballoons in the injectable liquidcarrier, are amphipatic compounds like the phospholipids; they alsoincrease water permeability and rate of biodegradability. The quantityof additives to be incorporated in the polymer forming the membrane ofthe present microballoons is extremely variable and depends on theneeds. In some cases no additive is used at all, in other cases amountsof additives, which may reach about 20% by weight of the polymer, arepossible.

The third category of stabilized or encapsulated gas bubbles aremicroparticles, suspensions of porous particles of polymers or othersolids, which carry gas microbubbles, entrapped within the pores of themicroparticles. These systems, which include aggregates ofmicroparticles, have gas contained therein or otherwise associatedtherewith (for example being adsorbed on the surface thereof and/orcontained within voids, cavities or pores therein, e.g. as described inEP 0122 624, EP 0123 235, EP 0365 467, U.S. Pat. Nos. 5,558,857,5,607,661, 5,637,289, 5,558,85, 5,137,928, WO 9521631 or WO 93/3809,incorporated by reference herein in their entirety.

The encapsulated or stabilised gas bubbles (stabilized microbubble,microballoon or microparticle suspensions) used in the present inventionmay conveniently be administered in a pharmaceutically acceptableaqueous liquid carrier. Suitable liquid carriers are water, aqueoussolutions such as saline (which may advantageously be balanced so thatthe final product for injection is not hypotonic), or solutions of oneor more tonicity adjusting substances such as salts or sugars, sugaralcohols, glycols and other non-ionic polyol materials (e.g. glucose,sucrose, sorbitol, mannitol, glycerol, polyethylene glycols, propyleneglycols and the like). In practice all injectable compositions shouldalso be as far as possible isotonic with blood. Hence, before injection,small amounts of isotonic agents may also be added to the suspensions ofthe invention. The isotonic agents are physiological solutions commonlyused in medicine and they comprise aqueous saline solution (0.9% NaCl),2,6% glycerol solution, 5% dextrose solution, etc.

Other excipients may if desired be present in the composition beingdried or may be added on formulation for administration. Such excipientsmay for example include pH regulators, osmolality adjusters, viscosityenhancers, emulsifiers, bulking agents, etc. and may be used inconventional amounts.

The encapsulated or stabilised gas bubbles are filled with a gas or agas mixture comprising a physiologically acceptable gas selected fromthe group consisting of fluorinated gases, including sulfurhexafluoride, trifluoromethylsulfur pentafluoride, Freons® (e.g. organiccompounds containing one or more carbon atoms and fluorine such as CF₄,CBrF₃, C₄F₈, CClF₃, CCl₂F₂, C₂F₆, C₂ClF₅, CBrClF₂, CBr₂F₂, C₃F₈ andC₄F₁₀ and mixtures thereof), and perfluorocarbons; air; nitrogen;oxygen; carbon dioxide; hydrogen; nitrous oxide; inert gases such ashelium, krypton, xenon, and argon; hyperpolarized gases; a low molecularweight hydrocarbon (e.g. containing up to 7 carbon atoms), for examplean alkane such as methane, ethane, a propane, a butane or a pentane, acycloalkane such as cyclobutane or cyclopentane, an alkene such aspropene or a butene, or an alkyne such as acetylene; an ether; a ketone;an ester; a halogenated low molecular weight hydrocarbon (e.g.containing up to 7 carbon atoms); or a mixture of any of the foregoing.At least some of the halogen atoms in halogenated gases advantageouslyare fluorine atoms.

Biocompatible halogenated hydrocarbon gases may, for example, beselected from bromochlorodifluoromethane, chlorodifluoromethane,dichlorodifluoromethane, bromotrifluoromethane, chlorotrifluoromethane,chloropentafluoroethane, dichlorotetrafluoroethane and perfluorocarbons,e.g. perfluoroalkanes such as perfluoromethane, perfluoroethane,perfluoropropanes, perfluorobutanes (e.g. perfluoro-n-butane, optionallyin admixture with other isomers such as perfluoro-isobutane),perfluoropentanes, perfluorohexanes and perfluoroheptanes,pefluorooctanes, perfluorononanes, perfluorodecanes; perfluoroalkenessuch as perfluoropropene, perfluorobutenes (e.g. perfluorobut-2ene) andperfluorobutadiene; perfluoroalkynes such as perfluorobut-2-yne; andperfluorocycloalkanes such as perfluorocyclobutane,perfluoromethylcyclobutane, perfluorodimethylcyclobutanes,perfluorotrimethylcyclobutanes, perfluorocyclopentane,perfluoromethylcyclopentane, perfluorodimethylcyclopentanes,perfluorocyclohexane, perfluoromethylcyclohexane andperfluorocycloheptane. Other halogenated gases include fluorinated, e.g.perfluorinated, ketones such as perfluoroacetone and fluorinated, e.g.perfluorinated, ethers such as perfluorodiethyl ether. Contrast agentscontaining sulphur hexafluoride, perfluorocarbons, such as for example,perfluoropropane or perfluorobutane or mixtures thereof with air,oxygen, nitrogen, helium or CO₂ are preferred, with SF₆ and C₄F₁₀ areparticularly preferred.

The gas can be a mixture of the gases above defined. In particular thefollowing combinations are particularly preferred: a mixture of gases(A) and (B) in which, at least one of the gases (B), present in anamount of between 0.5-41% by vol., has a molecular weight greater than80 daltons and (B) is selected from the group consisting of SF₆, CF₄,C₂F₆, C₂F₈, C₃F₆, C₃F₈, C₄F₆, C₄F₈, C₄F₁₀, C₅F₁₀, C₅F₁₂ and mixturesthereof and (A) is selected from the group consisting of air, oxygen,nitrogen, carbon dioxide and mixtures thereof the balance of the mixturebeing gas A.

In addition, as discussed, the contrast agent may include a gasprecursor (e.g. a compound or compound mixture which is partially ingaseous form (including vapour) at normal human body temperatures (37°C.) i.e. C₅F₁₂, C₆F₁₄, cyclohexane, cyclooctane, hexane, cyclopentane,etc.).

Particularly preferred are gas precursors with boiling points between 20and 80° C. The gas precursor may be used alone or in combination with agas or another gas precursor.

The foregoing description will be more fully understood with referenceto the following Examples. Such Examples, are, however, exemplary ofmethods of practising the present invention and are not intended tolimit the scope of the invention.

EXAMPLES Example 1

FIG. 4 shows the sub- and ultraharmonic energy curves (top and bottom,respectively) as a function of time for an air bubble with a radius of2.2 μm as obtained by computer simulation. The curves were calculated,as described in the specification, for different values of thesurrounding liquid pressure. The liquid pressure is indicated by theoverpressure, i.e. the pressure value over the atmospheric pressure of760 mmHg. The values for the overpressure were 0, 50, 100 and 200 mmHg.The different lines show the respective energy curves. From these curvesthe mean response time was calculated according to equation (2) and thevalues for the mean sub- and ultraharmonic response time are given intable 1 in the third and fourth column, respectively. As a reference,the time for the air bubble to completely disappear as a function of theoverpressure is calculated by equation (1), and is given in the secondcolumn. By looking at the difference in sub- and ultraharmonic responsetime (column 3 and 4) for different pressures (difference between therows), it is clear that with the new method similar or highersensitivity can be obtained than by measuring the disappearance time.Additionally, a significant advantage of the new method is that theacquisition time will be much shorter (2-3 times) compared to theacquisition time for measuring the total disappearance time (column 2),which is important for real time application of the method.

TABLE 1 Disappearance time, td, mean response times for subharmonic,t_sub, and ultraharmonic, t_ul, as a function of the overpressure. Pov[mmHg] td [ms] t_sub [ms] t_ul [ms] 0 120.8 51.0 51.5 50 112.7 42.6 43.1100 105.7 35.7 36.4 200 94.1 26.7 27.0

Example 2

This example shows the sensitivity of the new method to measure clinicalrelevant pressure differences of 10 mmHg, for an air bubble with aradius of 2.2 μm as obtained by computer simulation. The results of thisexample are shown in table 2. The overpressures range from 80 to 120mmHg in steps of 10 mmHg. The difference in disappearance time (column 2in table 2) ranges from 1.2 to 1.4 ms per 10 mmHg of pressure change.The difference in mean response time for the subharmonic (column 3 intable 2) ranges from 1.7 to 2.7 ms. The difference in mean response timefor the ultraharmonic (column 4 in table 2) ranges from 2.1 to 2.8 ms.This means that by using the new method, the sensitivity increased by40-100%, compared to methods that consider the complete disappearance ofgas bubbles.

TABLE 2 Disappearance time, td, mean response times for subharmonic,t_sub, and ultraharmonic, t_ul, as a function of the overpressure. Pov[mmHg] td [ms] t_sub [ms] t_ul [ms] 80 108.4 40.2 41.3 90 107.0 37.538.5 100 105.7 35.7 36.4 110 104.4 34.0 34.0 120 103.2 32.1 31.5

1. A noninvasive measuring method for remotely determining localphysical parameters of a fluid-filled cavity, based on the combined useof encapsulated or stabilized gas bubbles and ultrasound waves,comprising the steps of: a) administering encapsulated or stabilized gasbubbles to a fluid filled cavity; b) applying a first ultrasound pulsedwave or a train of pulsed waves to the fluid tilled cavity to destroythe encapsulated or stabilized gas bubbles and generate free-gasbubbles; c) applying a second ultrasound pulsed wave or train of pulsedwaves at a frequency chosen for exciting the sub- and/or ultraharmonicresponse of the free-gas bubbles; d) determining the mean sub- and/orultraharmonic response time corresponding to the mean time for sub- orultraharmonics to appear after the generation of the free gas bubblesfrom the encapsulated or stabilized bubbles; and e) determining a valuefor a local physical parameter on the basis of the response time of stepd).
 2. The measuring method according to claim 1, characterised in thatthe first ultrasound pulsed wave or train of pulsed waves is tuned insuch a way that the size of the released free-gas bubbles is larger thanthe subharmonic size.
 3. The measuring method according to claim 1 or 2,characterised in that the frequency and amplitude of the firstultrasound pulsed wave or train of pulsed waves can be chosenindependently of the second ultrasound pulsed wave or train of pulsedwaves.
 4. The measuring method according to claim 1, characterised inthat the frequency and amplitude of the second ultrasound pulsed wave ortrain of pulsed waves can be chosen and independently of the firstultrasound pulsed wave or train of pulsed waves.
 5. The measuring methodaccording to any one of the claims 1 or 2, wherein the local parametersare selected from the group consisting of the pressure, the temperatureand the gas concentration.
 6. The measuring method according to claim 1,wherein the encapsulated or stabilized gas bubbles are selected from thegroup consisting of microbubbles bounded by a very thin envelopeinvolving the surfactant bound at the gas to liquid interface,microballoons bounded by a material envelope made of organic polymers orbiodegradable water insoluble and at room temperature solid lipids andmicroparticles of polymers or other solids, which carry gasmicrobubbles, entrapped within or associated with the pores of themicroparticles.
 7. The measuring method according to claim 6, in whichthe encapsulated or stabilised gas bubbles comprise gas or a gas mixtureselected from the group consisting of sulfur hexafluoride, aperfluorocarbon, air, nitrogen, carbon dioxide, helium, krypton, xenon,argon, methane, hyperpolarized helium, hyperpolarized xenon and mixturesthereof.
 8. The measuring method according to claim 7, in which theencapsulated or stabilized gas bubbles comprise a perfluorocarbonselected from the group consisting of perfluoromethane, perfluoroethane,perfluoropropane, perfluorobutane, perfluorocyclobutane,perfluoropentane, perfluorohexane and mixtures thereof.
 9. The measuringmethod according to claim 6, wherein at least one of the surfactantscomprises a film-forming phospholipid.
 10. The measuring methodaccording to claim 9, wherein the phospholipid film forming surfactantis selected from the group consisting of a saturated phospholipid, asynthetic non-saturated phospholipid and mixtures thereof.
 11. Themeasuring method according to claim 10, wherein the phospholipid filmforming surfactant is a saturated phospholipid selected from the groupconsisting of saturated phosphatidic acid, saturatedphosphatidylcholine, saturated phosphatidyl-ethanolamine, saturatedphosphatidylserine, saturated phosphatidylglycerol, saturatedphosphatidyl-inositol, cardiolipin and sphingomyelin.
 12. The measuringmethod according to claim 6, which the encapsulated or stabilized gasbubble is a microballoon bounded by a polymer selected from the groupconsisting of polylactic or polyglycolic acid and their copolymers,denaturated serum albumin, denaturated haemoglobin, polycyanoacrylate,and esters of polyglutamic and polyaspartic acids or a biodegradablewater insoluble and at room temperature solid lipid selected from amono-, di- or tri-glycerides, fatty acids, sterols, waxes and themixtures thereof.
 13. The measuring method according to claim 12, inwhich the microballoon is bounded by a saturated tri-glycerides selectedfrom the group consisting of tristearine, tripalmitine or mixturesthereof with other glycerides.
 14. A method of diagnostic ultrasound fordetermining local physical parameters of a fluid-filled cavity “in situ”which comprises the steps of: a) administering to a subject a fluidagent containing encapsulated or stabilized gas bubbles; b) applying afirst ultrasound pulsed wave or a train of pulsed waves to thefluid-filled cavity to destroy the encapsulated or stabilized gasbubbles and generate free-gas bubbles; c) applying a second ultrasoundpulsed wave or train of pulsed waves around a frequency specificallychosen for exciting the sub- and/or ultraharmonic response of thefree-gas bubbles; d) determining the mean sub- and/or ultraharmonicresponse time corresponding to the mean time for sub- or ultraharmonicsto appear after the generation of the free gas bubbles from theencapsulated or stabilized bubbles; and e) determining a value for alocal physical parameter on the basis of the response time of step d).15. The method according to claim 14, wherein said subject is avertebrate and said fluid agent containing encapsulated or stabilisedgas bubbles is introduced into the vasculature or into a body cavity ofsaid vertebrate.